In Vitro Cell. Dev. Biol.Plant 41:113123, March April 2005 q 2005 Society for In Vitro Biology 1054-5476/05 $18.00+0.

00

DOI: 10.1079/IVP2004619

RNA SILENCING IN PLANTS

ESRA GALUN*

Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76 100, Israel
(Received 5 November 2004; accepted 16 November 2004; editor E. C. Pua)

Summary The notion that the introduction of alien RNA into an organism can cause the silencing of endogenous genes and transgenes came to light in plants during the last decades of the 20th century. It was based on revealing virus-induced gene silencing (VIGS) and on the protection against pathogenic viruses by pre-infection with less pathogenic plant viruses or components of such viruses as well as on co-suppression phenomena. The breakthrough in RNA silencing research was the discovery of Mello, Fire and associates that double-stranded RNAs (dsRNAs) can silence specically homologous genes in the nematode Caenorhabditis elegans. The discovery in C. elegans, published in 1998, immediately initiated studies in protozoa, metazoa, fungi, and plants, and similar RNA silencing mechanisms, albeit with some notable differences, were subsequently revealed in almost all eukaryotic organisms in which they were looked for. Investigators dealing with the different organisms were well aware of each others results and a very active eld of study emerged within a few years. Investigators of plant RNA silencing beneted from the ndings in other organisms, especially in C. elegans, Drosophila, and mammals, where the protein complexes involved in RNA silencing, such as the Dicer complex and the RNA-induced silencing complex (RISC), were studied intensively. The study of RNA silencing in plants followed two avenues. In one avenue the process of initiation of endogenous dsRNA was followed, also the fate and the impact of dsRNA that was introduced into plant cells was investigated. It was found how this dsRNA is cut into , 21 nt fragments and the derived ssRNA of ,21 nt may guide the RISC to cleave specic mRNA sequences. In the other avenue the formation of hairpin, or stem loop RNA sequences, from transcripts of genomic sequences, was investigated. The maturation of these RNA structures into mature microRNA was studied and the possible roles of endogenously formed and introduced microRNAs in the regulation of expression of plant genes were gradually revealed. This review will update the ndings in these two avenues. Key words: angiosperms; Arabidopsis; co-suppression; Nicotiana; post-transcriptional gene silencing (PTGS); transcriptional gene silencing (TGS); virus-induced gene silencing (VIGS).

Historical Background The term RNA silencing stands for mechanisms by which RNA sequences (that are not mRNAs, tRNAs, or ribosomal RNAs) regulate gene expression. Gene expression by itself may have either or both of the following meanings. This term is used to describe the reduced level of transcription of mRNA from genomic DNA and/or for describing the reduced levels of the ultimate gene products, usually protein, from specic genes. Since the end of the rst half of the 20th century it has been established that in cellular organisms, the genetic information is stored in the DNA, of eurkaryotic organisms, in linear sequences. This one-dimensional organized information is placed, in eukaryotic organisms, in the nucleuslocated chromosomes as well as in the cellular organelles: mitochondria and (in plants) plastids. It became clear that not all the genes in the nucleus or in the organelles are constantly expressed. On the contrary, only a small part of the genes are expressed in a given cell and in a dened period. How does the cell
*Author to whom correspondence should be addressed: Email esra.galun@ weizmann.ac.il

regulate the expression of genes? Studies in prokaryotes (bacteria) (e.g., Jacob and Monod, 1959) revealed the regulatory capacity of a cis element of the transcript: the promoter. It then became evident that a plethora of additional regulatory mechanisms are involved in gene expression in eukaryotes. These mechanisms include those that are encoded by the gene itself (cis-regulating sequences), in the stability of the mRNA, as well as mechanisms involved in the chromatin remodeling. That chromatin has a role in the regulation of gene expression in eukaryotic cells was inferred in the early 1960s by James Bonner and associates, who studied chemical changes in the chromatin of peas that could be implicated in the regulation of gene expression (e.g., Bonner et al., 1961; Huang and Bonner, 1962). Among the many other means by which the cell regulates the level of gene expression is the existence of multiple copies of the same gene. The latter means is represented in the genes that encode ribosomal RNAs. Could there be a completely different means to regulate (negatively) the expression of specic genes? Such a negative regulation could operate at the transcription level, at the post-transcription level or at the level of translating the transcript (mRNA) into protein. Clues for the involvement of RNA sequences, in these additional means of negative regulation of gene expression, 113

114

GALUN

emerged in plants in the 1980s. I shall briey survey these clues below. Early clues for RNA silencing in plants. In my recent book (Galun, 2005) I provided ample information on the prehistory and history of gene silencing, with emphasis on angiosperm plants. Here I shall note only a few cases of gene silencing that were reported before it was proven that these silencings are involved with doublestranded RNA (dsRNA). Two elds of investigation shall be mentioned: co-suppression and cross-protection by viruses. The term co-suppression was assigned to the phenomenon that occurred when a transgene was introduced into the genome of an organism, causing a reduced expression of the transgene that was introduced, as well as a reduced expression of the homologous (or similar) endogenous gene. Early cases of co-suppression were revealed when genetic transformation of plants became an established procedure, commonly executed by Agrobacteriummediated transformation of plants (see Galun and Breiman, 1997, for full coverage of this subject). It was frequently observed that when a transgene was introduced it did not enhance the expression of the endogenous gene, but rather, on the contrary, the expressions of both the endogenous and the transgene were silenced. This cosuppression was not recorded in all cases, but it happened occasionally and not in all the transformed progeny. The reason for that silencing was enigmatic for several years. Two publications in which co-suppression was described in detail and in which the cosuppression had clear (and colorful) manifestations appeared, in tandem, in the journal Plant Cell: Napoli et al. (1990) and van der Krol et al. (1990). Both publications were on the pigmentation of petunia petals and in both studies the initial intention was to enhance the pigmentation, but the authors of both articles were surprised by the results of reduced pigmentation. It should be noted that an early key compound, in the metabolic pathway of petal pigmentation, is chalcone. This is a condensation product of one 4-coumaryol-CoA molecule with three molecules of melanoyl-CoA. The condensation is performed by the enzyme chalcone synthase (CHS). In further metabolic steps the colorless dihydroavonol is produced. The latter is processed further by the enzyme dihydroavonol reductase (DFR), leading, nally, to the pigmented anthocyanin glycosides. In petunia CHS is rate-limiting for pigmentation. Napoli et al. (1990) focused on CHS. To over-express this gene they genetically transformed petunia plants with a chimeric construct that included the 35S promoter (of CaMV) and the coding sequence for CHS. This chimeric construct was introduced into the genomes of several petunia varieties by Agrobacterium-mediated transformation. This resulted in many transgenic petunia plants that had white owers or owers that had patterns of white and pigmented owers. The white petals had a 50-fold lower mRNA for CHS than the wild-type pigmented petals. Some petals reverted to full pigmentation and the reverted petals had the normal level of CHS mRNA. Hence the introduction of a transgene could clearly cause the co-suppression of the expression of CHS. A similar phenomenon was revealed by van der Krol et al. (1990) who studied the effect of the CHS transgene as well as that of the DFR transgene. In both cases co-suppression was observed. Several causes for the co-suppression were inferred but dsRNA was not yet suggested. M. A. Matzke and A. J. Matzke, then in Salzburg, were engaged in genetic transformation of tobacco. In one of their studies (Matzke et al., 1989) they analyzed the results of two sequential genetic transformations. In one transformation, with

T-DNA-I, the plasmid contained the coding sequence for kanamycin resistance and the code for the nopaline synthase reporter gene. The subsequent transformation was with the T-DNA-II plasmid that contained the code for hygromycin resistance and the code for the octopine synthase (as reporter gene). When tobacco plants were transformed sequentially, rst with the T-DNA-I plasmid and then with the T-DNA-II plasmid, the expression of the T-DNA-I-encoded sequences was silenced. Was dsRNA, that could result from the similarity of promoters in the two types of plasmids, produced in the double-transformed plants? What the authors did reveal was that the promoters of the T-DNA-I were strongly methylated by the subsequent transformation with T-DNA-II. Both this methylation and the silencing of T-DNA-I were (fully or partially) removed in subsequent progenies after self-pollination or outcrossing, when the T-DNA-II sequence was eliminated from the genome of the respective plants. Was this methylation triggered by dsRNAs? There was no answer to this question as the question was not asked at the time by these authors. Evidence in favor of the notion that de novo methylation of genomic DNA can be mediated by RNA came only several years later from a study by Wassenegger et al. (1994). These are merely a few examples of co-suppression after genetic transformation. In many cases the investigators found a great variability in the transformed plants, from no expression of the transgenes up to rather high expression of these genes. Because the investigators were interested only in transformants with high transgene expression, the transformants with low transgene expression were just ignored. An authoritative and excellent review on co-suppression in plants was provided by Richard Jorgensen (2003). Virus-induced gene silencing (VIGS). Most plant viruses have an RNA genome. The awareness that plants may be immunized against severe viral pathogens by rst infecting the plants with a related, but much less virulent virus or with its RNA genome, is probably rather old. Although there are no precise records for the beginning of this awareness, such immunizations were practiced with notable success in the Citrus industry. In specic cases Citrus orchards were infected with a benign virus and two goals were achieved: the infection stunted the trees, simplifying their management, and also the danger of further severe viral infection was reduced or even eliminated. Milton Zaitlin and associates of the Cornell University (Golemboski et al., 1990) found that when a part of a cDNA from the genome of tobacco mosaic virus (TMV) strain U1 was introduced into tobacco plants, by Agrobacterium-mediated genetic transformation, these plants became resistant to TMV U1. Further information on a specic immunization against viral infection came from D. C. Baulcombe and associates of the John Innes Centre near Norwich (UK). Longstaff et al. (1993) found that when they mutated, in potato virus X (PVX), the gene that encodes the viral replicase, the respective PVX mutants were non-infectious in tobacco plants or in their protoplasts. But when viral-genome fragments, that contained the mutated codes, were used to transform tobacco plants and these plants were then infected with wild-type PVX, the infection caused a very low level of viral RNA in the infected cells and no viral RNA in systemic leaves (that were not infected directly). Other investigators reported similar results (e.g., de Haan et al., 1992; Lindbo et al., 1993; Smith et al., 1994). It was thus suggested that the resistance to viral infection was correlated with post-transcriptional suppression of transgene mRNA accumulation. A further study of the Baulcombe laboratory (Mueller et al.,

RNA SILENCING IN PLANTS

115

1995) indicated that the viral RNA polymerase (RdRP) is essential for the infection of plants by PVX, suggesting that gene silencing and resistance to viral infection involve the same mechanisms. These investigations as well as additional studies, in this line, were reviewed by Baulcombe and English (1996) and by English et al. (1996). The Baulcombe laboratory came up with several suggestions regarding VIGS that were later veried when the role of RNA degradation, imposed by homologous (double-stranded) RNA, was established. This laboratory also reported the traveling of the resistance from lower, infected leaves, to systemic leaves that are higher in the plant. The Baulcombe laboratory suggested that VIGS involves mRNA degradation and reported the existence of small RNA fragments during the VIGS process. On the other hand, several hypotheses on silencing such as the involvement of specic proteins, the effect of a threshold level of RNA, and the role of aberrant RNA in gene silencing were not substantiated. It was not only the Baulcombe laboratory that came rather close to revealing the real basis of RNA-induced gene silencing. Richard B. Flavell and associates (e.g., Metzlaff et al., 1997), also from the John Innes Centre, approached this silencing from a different angle. This team also studied the silencing of the CHS gene in transgenic petunia plants and concluded that the post-transcriptional gene silencing (PTGS) goes through a step of RNA RNA hybridization, meaning the formation of dsRNA. But neither the laboratory of Baulcombe nor the laboratory of Flavell put their idea to experimental test: introducing into plants sequences of specic dsRNA that are homologous to certain genes and comparing the results with those that follow the introduction of single-stranded RNAs (either sense or antisense) that have the same nucleotide sequences. From 1997 the eld of studying VIGS became very active and it was not only aimed at protecting plants from virus infection, but also as a more general tool to silence specic genes. Such studies were conducted even before the mechanism of VIGS was fully understood (e.g., Depicker and Van Montagu, 1997). The Baulcombe laboratory developed an experimental procedure (Ruiz et al., 1998) in which the PVX genome was manipulated to contain (and express) a transgene. The PVX thus served as a Trojan horse in this procedure. By inserting, into an engineered PVX, a sequence of the phytoene desaturase (PDS) gene they could silence the endogenous PDS, rendering infected Nicotiana benthamiana plants extremely sensitive to photo-bleaching. Interestingly, there was no difference in the silencing whether the PDS code was inserted into the PVX in either the sense (i.e., PDS) or in the antisense (i.e., SDP) direction. Similar silencing was also observed in transgenic N. benthamiana plants that expressed a GFP (green uorescent protein) gene, and were transformed again with PVX containing a truncated GFP gene. The truncated GFP sequence caused silencing of the GFP. The investigators found that the process of silencing was rather elaborated in the developing plants. Voinnet and Baulcombe (1997) showed that GFP silencing could be achieved also by Agrobacterium-mediated transformation (rather than by infection with engineered PVX). These studies clearly indicated that the silencing-signal can move considerable distances in the plant. Moreover, the silencing signal is capable of movement, in the plants, even across grafts. This latter nding was reported by Palauqui et al. (1997) of the laboratory of H. Vaucheret, in INRA, Versailles, France. As for protecting plants against viral infection, it was clear that both the virus as well as the silencing signal move systemically in

the plants. In cases where the silencing-signal moves ahead of the virus the plant may recover from the viral symptoms. Evidently there is a war going on between the plant and their viral foes. As shall be indicated below, there is an additional component of this war: viral suppression of RNA silencing. Externally Induced Silencing Revealing components of RNA silencing and silencing plant genes. As indicated in the Historical Background of this review, early investigations on gene silencing were conducted in plants, where the processes of VIGS and co-suppression were studied intensively during the later years of the 20th century. In some studies the investigators came rather close to revealing the actual trigger of RNA silencing: the dsRNA. But the denite proof for the role of short fragments of dsRNA in RNA silencing came from the now classical study of Craig Mello, Andres Fire and associates (Fire et al., 1998) with the nematode Caenorhabditis elegans. These investigators injected ssRNA (sense or antisense) or dsRNA, homologous to specic genes and found that the ssRNA had almost no effect on the expression of these genes while the injection of a few molecules of dsRNA silenced the expression of the homologous gene in the worms and this silencing was even transmitted to the sexual progeny. The latter transmission clearly suggested that there was a catalytic or amplication component in this interfering process. The interference process was then termed RNAi. The Fire et al. (1998) publication immediately sparked attempts to silence genes through the RNAi procedure in protozoa, fungi, lower metazoa, insects, and vertebrate animals, as detailed by Galun (2005). Obviously, plants were not excluded from these attempts. Hamilton and Baulcombe (1999) rationalized that if dsRNA is the trigger for PTGS, it is plausible that antisense mRNA hybridizes with sense mRNA, causing the formation of dsRNA. These investigators did not nd long dsRNA in their experimental system but did reveal short (, 25 nt) RNA species, that had homology with the silenced gene. The experimental system included transgenic tomato plants in which the ACO gene, that encodes 1-aminocyclopropane-1-carboxylate oxidase, was silenced by PTGS. Similar ndings were observed in transgenic tobacco plants that contained a b-glucuronidase (GUS) transgene that was then silenced by PTGS and in N. benthamiana plants in which a GFP was silenced. In all these cases there were , 25 nt RNA fragments that could be revealed by a sense probe from the respective mRNA. Were these fragments ssRNA or dsRNA? There was no clear answer to this question. Important information on RNA silencing came from the laboratory of Peter Waterhouse in Australia (e.g., Waterhouse et al., 1998; Smith et al., 2000). This laboratory previously conducted anti-viral studies in plants and very soon after the Fire et al. (1998) publication turned their attention to RNAi in plants. They revealed that in plants, just as in nematodes, dsRNA is far more efcient at gene-silencing than ssRNA. The Waterhouse team used several kinds of RNA constructs to reveal which are the most efcient for gene silencing. For example, they found that a construct that can fold into a stem-loop conguration and thus had dsRNA stems could cause 100% silencing of a desaturase gene (that has homology with the applied construct) in Arabidopsis and by that suppress the conversion of oleic acid to linoleic acid. This overall approach, to introduce dsRNAs with a length of 50 nt or more, into plants in order to silence specically genes that

116

GALUN

encode mRNA having homology to these dsRNA, became fashionable during 1999, 2000, and 2001. It has to be noted that in mammals the introduction of long fragments of dsRNA (longer than about 30 nt) is problematic in mature tissues. Such long dsRNAs induce a non-specic inhibition of gene expression. No such non-specic inhibition was observed in plants. On the other hand, the experimental procedure of introducing the appropriate silencing dsRNAs into plant cells is much more problematic than in nematodes. In the latter worms, such dsRNAs can be introduced by injection, by feeding on bacteria that produce the dsRNA, or even by soaking the worms in a solution that contains the dsRNA. Agrobacterium-mediated genetic transformation is a relatively simple procedure in solanacean plants such as tobacco, tomato, and potato; less simple in plants of other dicot families; and quite difcult in plants of the Gramineae family, such as wheat, barley, and maize (see Galun and Breiman, 1997; Galun and Galun, 2001). A particle bombardment system is applicable in the latter crop plants and this procedure is rather efcient in the introduction of nucleic acids for transient gene expression. Thus Schweizer et al. (2000) of the Institute of Plant Biology, Zurich, Switzerland, bombarded cereal leaves with tungsten particles that were coated with supercoiled reporter plasmids and dsRNAs. The reporter genes (for GUS or GFP) could thus be expressed in the leaves and could also be silenced by co-bombardment with the appropriate dsRNA. Similarly, dsRNA could silence, in barley and in maize, the gene for dihydro-avonol-4-reductase. Furthermore, an appropriate dsRNA could reduce the expression-level of a gene of the pathogenic fungus Blumeria graminis, that is implicated in pathogenicity, so that when the dsRNA was applied about 40 h before fungal infection, the infectivity was markedly reduced. By the year 2000 it was clear, through studies in several organisms (mainly nematodes, Drosophila, and mammalian cells) that RNA silencing (or RNAi) is a widespread mechanism that emerged early in the evolution of eukaryotes (see Bass, 2000 for an update of information available by that year). It became clear that an enzyme complex, termed Dicer, is targeted specically to dsRNA. Dicer has two catalytic sites of an RNase III-family type that cleave dsRNA at two locations that are separated by about 21 nt. The resulting dsRNA fragment has typically an overhang of one or two nucleotides at the 30 end and a phosphate at the 50 end. This fragment is separated into single-strand RNAs and one strand then serves as a guide for another protein complex, termed RISC (for RNA-induced silencing complex). The RISC is thus attached to a specic (homologous or homeologous) sequence in a mRNA. The mRNA is then either cleaved or its translation is suppressed (see Fig. 1 for a scheme of RNA silencing in plants). The details of the RNAi system became clearer in subsequent years and this system was found to be different, in some details, in plants from that of Drosophila and mammals. For example, in the latter organisms there is probably no amplication of the short interfering RNA (siRNA) while such an amplication does exist in plants and in nematodes. Moreover, studies in plants, such of those of Vaucheret and

associates in Versailles (e.g., Fagard et al., 2000) provided a signicant contribution to the mechanism of RNA silencing. These studies revealed several proteins that are required for this mechanism in plants (e.g., SGS2, QDE-1, EGO-1, AGO1). Differences were also detected between the specic roles of these proteins in plants versus other organisms. Also the number of family members and the respective genes that encode these proteins may be different in plants versus other organisms. For example, there are seven members of the EGO-1/SGS2 family in Arabidopsis and four such members in nematodes. Tang et al. (2003) of the Zamore laboratory developed an in vitro system in plants by which specic steps of the RNA silencing process could be analyzed. I shall not detail this and other studies of this kind as they all led to the current scheme of plant RNA silencing as shown in Fig. 1. The overall RNAi mechanism in plants was known in 2000 at a sufcient level to furnish a basis for the utilization of RNAi in the silencing of specic plant genes. Mentioning all the studies on experimentally silencing specic plant genes by dsRNA is beyond the scope of this review. Readers interested in these studies are referred to Chapter 11 of RNA Silencing (Galun, 2005) and an excellent recent review was provided by Meister and Tuschl (2004). Tools for RNA silencing in angiosperm plants. In previous sections of this review, I mentioned some of the tools for RNA silencing in plants. In summary, the rst and most direct means to cause this silencing is to synthesize short dsRNA, of about 21 nt. This dsRNA should have a 1 nt overhang at the 30 end and a phosphate at the 50 end. Such a dsRNA fragment should cause, in the plants cytoplasm, cleavage of mRNA at sites that are homologous to the synthesized dsRNA. While in nematodes and in certain animal tissues injection is the delivery of choice, in plants the bombardment of leaves with dsRNA-coated (tungsten or gold) particles is more efcient. The delivery by bombardment is obviously transient. I also mentioned above the genetic engineering of plant viruses, so that plants infected with the modied virus (e.g., modied PVX) will furnish a constant source of dsRNA that will even be replicated, together with the virus, in the infected plants. This led to a general delivery procedure termed by the Baulcombe laboratory as the Amplicon system. Another general procedure to furnish dsRNA is to integrate the code for this dsRNA into the plant genome. For that a tail-to-tail, inverted DNA sequence, has to be integrated. In this sequence there shall be rst a code that is homologous to the sense sequence of the respective dsRNA, then a spacer, and after the spaces the antisense (inverted) homologous sequence. Elements for (controlled or constitutive) promotion of transcription of the tail-to-tail DNA sequence should be added and the construct should be included in a plasmid that will be delivered to the plant. The delivery can be performed by Agrobacteriummediated genetic transformation. The transcript of the tail-to-tail construct is expected to fold into a hairpin (or stem-loop) RNA sequence. Plant cells know how to handle such hairpin RNAs because similar structures are produced in plants during the maturation of endogenous microRNAs. The endogenous microRNAs Q

FIG . 1. Generation of siRNA and microRNA in plants and their impact on gene expression. A, An overall view of the silencing process. B, A highly schematic cartoon of mRNA cleavage resulting from dsRNA. C, A highly schematic cartoon of the generation of microRNA that is bound to a RISC-like particle together with a compatible mRNA. This results in cleavage or the inhibition of translation from homologous or homeologous mRNA. Note that these cartoons are representing the presently assumed stages and future studies may change the models represented in these cartoons.

RNA SILENCING IN PLANTS

117

118

GALUN

of plants shall be handled in a later part of this review. The principal concept for the tail-to-tail DNA construct that will lead to a hairpin RNA and thus silence specic genes was elaborated by the laboratory of Waterhouse and associates (Wesley et al., 2001). These investigators provided detailed instructions on how to handle wide-range silencing through the construction of a standard vector that was termed pHANNIBAL. For RNA silencing, specic sequences can be integrated in the vector and the various pHANNIBAL-based vectors can then serve for specic silencing. The pHANNIBAL vectors have anking restriction sites, in order to plug-in the desired coding sequences. This procedure was further elaborated by the same laboratory (Stoutjesdijk et al., 2002; Helliwell and Waterhouse, 2003), using in addition to the pHANNIBAL vector also pKANKIBAL and pHELLSGATE vectors for successful, high-thoroughput, gene silencing in plants. Several additional means to improve the procedures for specic silencing of plant genes by the RNAi system were reviewed by Horiguchi (2004), who provided ample references on this subject. The construction of vectors for PTGS reached commercial companies as well as multinational collaborative efforts. For example, the Promega Corporation (www.promega.com) in its Promega Notes Magazine no. 87 featured instructions for RNAi. The Chang Bioscience Inc. offers a Support Vector Machine (SVM) that is an RNAi learning program in an electronic protocol book. Vectors intended for RNAi knockout (i.e., PTGS) in Arabidopsis were developed by the Agrikola project that is a European Unionsupported activity that provides detailed protocols (www.agriola.org). RNA silencing for crop improvement. The efcient application of gene silencing by RNA constructs obviously triggered investigators to use this approach for crop improvement. It is noteworthy to remember that crop plants in which RNA silencing was applied are genetically modied (GM) crops. Although the cultivation of GM crops such as maize, soybean, and cotton is constantly increasing, they are not universally accepted as food and/or feed crops. Thorough discussion of crop improvement and the manufacture of medical products by genetically engineered plants was provided by two books (Galun and Breiman, 1997; Galun and Galun, 2001). In this review I shall provide only a few examples of crop improvement by RNA silencing. A very good review, dealing mostly with the improvement of the nutritional value of crops, was recently published by Tang and Galili (2004). For some coffee consumers coffee beans with a low caffeine level is a desired product. True, there is a great variability in caffeine content among Caffea trees so that conventional breeding can lead to a low-caffeine cultivar. But it is a long process: it may take 25 yr. Ogita et al. (2003) of the Nara Institute of Science and Technology reported a shorter way: by RNA silencing. Three N-methyltransferase enzymes are involved in caffeine biosynthesis of Caffea trees. One of these is the threobromine synthase (CaMXMT1). The Japanese investigators focused on the gene that encodes this enzyme. They made constructs that, after transcription, should produce RNAi sequences that are homologous to the 30 untranslated region (30 UTR) of the CaMXMT1 mRNA, and by Agrobacteriummediated genetic transformation obtained young Caffea canephora plants. The leaves of the 1-yr-old transformed trees had reduced threobromine and caffeine contents. If this reduction is evident not only in the leaves but also in the future beans, and if such transformations are possible in C. arabica trees, this could lead to low-caffeine coffee beans.

Joachim Messing and associates of the Waksman Institute of Rutgers University intended to improve the nutritional value of maize-grain proteins by increasing the ratio between lysine and the other amino acids (Segal et al., 2003). There have been past efforts by corn breeders to elevate the relative level of lysine in maize grains by using a recessive mutant opaque-2. The level of lysine was consequently elevated but the respective breeding product had several drawbacks and it was not adopted by corn farmers. The idea of Segal et al. (2003) was to silence the gene that encodes a specic 22-kDa protein of the maize endosperm. The synthesis of the maize grain protein is rather elaborate so here are only some relevant features of it. The zeins of the maize-grain endosperm are classied into four sub-families: a, b, g, and d-zeins. Of these, a-zein accounts for 70% of the endosperm proteins. This latter zein is encoded by several genes, located on different chromosomes. Moreover, the a-zein was sub-divided into 19- and 22-kDa subfamilies. Reduction in the 22-kDa was expected to result in a better lysine content. Using an appropriate construct, that after transcription in the plant cells will form the desired hairpin RNA sequence that will be further processed into the required RNAi, the investigators were able to receive maize grains with an improved composition of amino acids. The vector for their transformation was introduced into the maize cells by particle bombardment that was followed by the regeneration of the desired transgenic maize plants. Gad Galili of the Weizmann Institute of Science is a veteran in the study of essential (e.g., lysine, threonine, methionine) amino acids in plants and recently suggested, in a joint publication with G. Tang (Tang and Galili, 2004), procedures to elevate these amino acids by RNA silencing. In the grains of rice the level of glutelin genes could be silenced in a study by Kusaba et al. (2003) in Japan, again changing the nutritional value of this crop. The last example of crop improvement by RNA silencing concerns the composition of oils in cotton seeds (Liu et al., 2002). Although cotton is considered a ber crop, cotton seeds are the worlds sixth largest source of vegetable oil. Paradoxically, while there is a shortage in the world of mineral oil, there is a world surplus of vegetable oils! The fatty acid composition of cotton seed has very little variation among existing cultivars with respect to palmitic, oleic, and lindeic acid (about 26%, 25%, and 58%, respectively). Changing these levels by breeding is therefore problematic. There are good reasons for changing the oil composition of cotton seeds for nutritional and industrial purposes. Liu et al. (2002) focused on two genes, ghSAD-1 and ghFAD2-1, that are D9-desaturase and D12-desaturase, respectively. The respective enzymes, encoded by these genes, convert stearic acid to oleic acid, and oleic acid to linoleic acid. These investigators synthesized constructs that would be transcribed into hairpinforming sequences that had homology (in their stems) to either the ghSAD-1 or the ghFAD2-1 gene. The appropriate vectors were used in Agrobacterium-mediated transformations and the respective transgenic cotton plants were obtained. The use of these hairpinforming constructs was rather effective. Transfer of the ghSAD-1 hairpin-forming construct increased the stearic acid content of the cotton seed oil from about 2.4% in a regular cultivar to about 40%; the silencing of the ghFAD2-1 gene increased oleic acid from 15% up to 77%. We should keep in mind that there are other means to change the oil composition of the respective crops (e.g., genetic transformation with a specic, alien enzyme-encoding gene, conventional breeding), but RNA silencing is also a plausible way.

RNA SILENCING IN PLANTS

119

Viral suppressors of RNA silencing. An apparently peaceful meadow is actually the arena of ferocious wars. Some of the wars are going on within the genomes of the plants: the attempts of transposable elements (TE) to colonize increasing spaces in the genomes of the plants and an active defense against the TE by the plants (see Galun, 2003). Another kind of war is between the plants and their pathogens. This later kind concerns our theme. The VIGS was mentioned above. The VIGS is clearly a way of the plant to defend itself against pathogenic invaders, especially viral pathogens. There were scholars who claimed that eukaryotes evolved the RNA-silencing mechanism as a way to improve their arsenal for their ght against TE and pathogens. As for viral pathogens, several of them have a way to ght back by suppressing the RNA silencing. Investigators became aware of viral suppression of RNA silencing many years ago. How many years is not clear to the present author. When Vicki Vance (1991) reported on the synergism between PVX and PVY (potato virus Y) mixed infection of tobacco plants, she noted previous observations that showed that in mixed infections by two unrelated plant viruses, the viruses may help each other and cause more severe disease symptoms than a single virus infection. It was then found (Vance et al., 1995) that this synergism was not limited to the PVX/PVY mixed infection. PVX also helped other viruses. Further studies of Vance, James Carrington (Texas A&M University), and associates indicated that the potyvirus genomes encode the broad-range pathogenicity enhancers. The history of revealing the viral genome-encoded polypeptides that counteract RNA silencing is beyond the scope of this review. A comprehensive coverage of this subject was provided by Roth et al. (2004). The latter review also provides ample references for the subject of viral suppressors of RNA silencing. By the time this review was submitted there was information on 16 such suppressors. Intensive studies have been conducted on some of these suppressors as the HC-Pro that is encoded by the potyviruses PVY and TEV (tobacco etch virus) and the p19 that is encoded by the genomes of TBSV (tomato bushy stunt virus) and other tombusviruses. These various suppressors not only vary with respect to their amino acid sequences but also with respect to their mode of suppression. Hence it is reasonable to claim that while RNA silencing had a monophyletic evolution, the various suppressors evolved independently from each other. As noted above, the mode of action of the suppressors differs considerably. For example, HC-Pro (i.e., of PVY) acts by blocking the maintenance of the PTGS mechanism in tissues where silencing had already been set; while another suppressor, the 2b of CMV (cucumber mosaic virus), rst reported by Ding et al. (1995, 2004), prevents the initiation of RNA silencing, but 2b does not suppress a PTGS that was already going on. A specic mode of action was recently attributed to a TYMV (turnip yellow mosaic virus) polypeptide termed p69. Studies aimed at providing more information on the mode of action of viral suppressors of RNA silencing are now going on. One example (see Zamore, 2004) is from P19. The structure of this polypeptide was analyzed by X-ray crystallography (Vargason et al., 2003; Ye et al., 2003). It appears that due to its structure P19 has a strong afnity to the short (, 21 nt) dsRNA that results from the fragmentation of long dsRNA by Dicer. P19 has a cradle into which the Dicerderived siRNA ts exactly. It is blind to the sequence of the nucleotides in this siRNA but does recognize its size and ends. Probably this binding interferes with the separation of the antisense

sequence from a given siRNA and thus prevents this antisense from becoming a guide to RISC. Hence, the suppressor interferes with the cleavage of the respective mRNA. Introducing P19 into plants, where it normally is not expressed, can cause severe morphological abnormalities. It was suggested that these abnormalities resulted from the suppression of the microRNAs of the new host of P19. As we shall mention below, microRNAs have vital roles in normal development, hence their suppression will abolish normal differentiation. While the viral suppressors probably evolved as a weapon by viruses in their war against their hosts, these suppressors should be taken into account by investigators. Such suppressors could be benecial when transgenes are to be expressed in transgenic organisms: the suppressor may prevent co-suppression. On the other hand, when RNAi procedures are applied to organisms which contain viral suppressors the latter may interfere with the expected RNA silencing. Transcriptional gene silencing (TGS). Gene silencing can also take place by the inhibition of gene transcription (TGS) through chromatin remodeling and DNA methylation. Moreover, these two processes could interact. A major kind of chromatin remodeling is by the methylation of lysine 9 (K9) in histone 3 (H3). This H3mK9 can cause a marked reduction of transcription and in plants it was assumed to interact with the methylation of cytosine in the DNA. Moreover, these two kinds of methylation are involved with RNA silencing. This is an emerging eld of investigation which occupied the team of Rob Martienssen in the Cold Spring Harbor Laboratory and other investigators. It was updated in a recent review (Lippman and Martienssen, 2004) that provides a thorough coverage of this subject with ample (100) references. I shall not detail this subject and readers who are interested in it are referred to the latter review. A special case of microRNA that is implicated with cytosine methylation will be presented below. Plant MicroRNA Description of terms. Several terms that are related to microRNA will be used in the following sections of this review. MicroRNA should be regarded as a concept rather than a dened term. The microRNA genes are sequences, in the eukaryotic genome, that have the capability to be converted, after due transcription and processing, into mature microRNA. The mature microRNAs are ssRNA sequences of about 21 nt that do not differ from the ssRNA derived from the siRNAs that were mentioned above, but for their source: they are derived from microRNA genes. After the microRNA gene is transcribed, the transcript (RNA) will fold into a stem-loop structure in which there is a loop on one side of a dsRNA stem and on the other side of the stem there are two unpaired ssRNA tails. This folded RNA is rst processed to the pri-microRNA, by the shortening of the tails, and by further processing the pre-microRNA is formed (see Fig. 1). One of the two strands of the stem, in the pre-microRNA, is separated to form the mature microRNA. The mature microRNA ranges in size from about 21 to 25 nt. It guides the RISC to a homologous mRNA. For brevity the microRNA genes were also termed miRNAs or miRs. Revealing plant microRNAs. Just as the rst understanding of the role of dsRNA in gene silencing resulted from studies in nematodes (Fire et al., 1998), so also the awareness of microRNAs came from studies in these worms. Two genes in C. elegans that

120

GALUN

were found to have a profound role in differentiation of the larvae of this worm, lin-4 and let-7, were found to be microRNA genes. They were encoded by tail-to-tail inverted sequences and their transcripts were found to be processed into mature microRNAs that guided the RISC-like particle (also termed miRNP) to specic mRNAs that will be silenced. By this the formation of specic proteins that are not required or even abolish developmental stages of the larvae, can be prevented (Reinhart et al., 2002). Further studies, especially in worms and Drosophila, clearly indicated that microRNAs play an evolutionary conserved role in development of metazoa. The microRNA genes exhibited temporal and/or tissuespecic patterns of gene expression and thus are essential for normal differentiation (see Meister and Tuschl, 2004 for review). Soon after the role of microRNA in animals was revealed, several teams from different laboratories submitted their respective reports on studies with plants: Llave et al. (2002a), Mette et al. (2002), Reinhart et al. (2002), and Park et al. (2002). These submissions were done in March, April, May, and June of 2002, respectively. Because the full genomic sequence of Arabidiopsis thaliana was available, the search for microRNA genes focused on this species. The coding sequences for plant microRNAs were found in different chromosomes. Some were found clustered in intergenic regions and others were located in introns of protein-coding genes, in transposon-like sequences as well as in or around the repeats that code for the structural 5S rRNA genes. They could be detected by computer programs and thereafter be veried by molecular techniques. The transcripts of these microRNA were of different lengths. They could be 200 nt long and were commonly longer than the transcripts of animal microRNA genes, but as the latter they could be folded into stem-loop structures. Such stem-loop structures are the pri-microRNAs that are further processed as shown schematically in Fig. 1. The microRNA genes were found also in monocots (rice) and interestingly some of these genes were rather conserved in Arabidopsis and rice that are considered to have their common ancestor about 250 million years ago. This was found by Reinhart et al. (2002) for miR162 and miR164. The conservation of sequences among miRs of related and distant plant species was helpful in identication of the miRs. Several important ndings were made during the early studies on plant microRNA. Llave et al. (2002b) found that the miR39 is homologous to several regions of the family of Scarecrow-like genes that are assumed to code for transcription factors involved in oral differentiation. If indeed miR39 is active in cleavage of the respective mRNAs of these factors, it is an important regulator of plant differentiation. It was also found that the Dicer-like protein of plants (DCL1) has a nuclear-localization signal and therefore probably has a role in processing the transcripts of the microRNA genes in the nuclei of plants. In animals such a role was assigned to a complex termed Drosha. It was also found that the pre-microRNAs are exported from the nucleus to the cytoplasm by a protein termed exportin but the detailed understanding of this process awaits further studies. After further processing, as indicated above, a short dsRNA is formed that is similar to that described above for virusinduced dsRNA: having a 1 or 2 nt overhang at the 30 end and a phosphate at the 50 end and a length of about 21 25 nt. This is the miRNA:miRNA* duplex, where miRNA means the antisense of its target sequence on a specic mRNA. The sense sequence, the microRNA*, is probably degraded. Details of microRNA processing and function were provided by Bartel and Bartel (2003) and Bartel

(2004). After the guiding (mature) ssmicroRNA is complexed with the miRNP and the combined particle is bound to the proper mRNA, either of two possibilities will follow: the mRNA may be cleaved or the translation from the mRNA will be stopped. Recently, a role in the methylation of a homologous DNA was also suggested (Bao et al., 2004). From studies with animal systems it was rst assumed that if there is not a full homology between the microRNA guide and a region in the mRNA target, translation will be inhibited, but if there is a full homology the mRNA will be cleaved. But then exceptions were revealed. In plants where commonly microRNAs were found to mediate the cleavage of mRNA, the inhibition of translation was also reported: miR 172 has a near-perfect homology to a region of its target (the mRNA for APETALA2), still miR172 causes inhibition of translation rather than degradation of the mRNA. Obviously the picture of microRNA processing and function is still far from being clear. Several additional components of the microRNA mechanism may be revealed in future studies. One example of nding such a component comes from the study of Nina Fedoroff and associates (Han et al., 2004) who studied mutants of the gene HYPONASTIC LEAVES1 (HYL1). The double mutant of hyl1 shows pleotropic effects on the morphology of Arabidopsis. In the presence of hyl1 the levels of three analyzed miRs (miR159, miR167, and miR171) are strongly reduced. It is therefore probable that the HYL1 protein has a role in the generation and processing of plant microRNAs. Plant microRNAs and their targets. The team of Bonnie and David Bartel was, as mentioned above (Reinhart et al., 2002), among the rst who revealed microRNAs in plants. Briey their method of identifying such miRs was to obtain small RNAs (of 18 26 nt) from Arabidopsis organs and to clone them. The clones were run on a gel and hybridized with antisense DNA of Arabidopsis and the hybridized fragments were used to screen the Arabidopsis genome. When homologous tail-to-tail sequences, on the genome, were found, this indicated coding sequences for miRs. Several of the suspected Arabidopsis miRs had matches in the rice genome. Then targets for these miRs were searched. In a further search a computer-assisted procedure was developed and many more plant miRs and some of their targets could be listed (Jones-Rhoades and Bartel, 2004). This could be only the tip of the iceberg because exposing plants to stress revealed additional miRs and targets for these miRs. As several teams hunted for miRs an agreement for a uniform microRNA annotation was reached (Ambros et al., 2003) and a microRNA repository/registry center was established at the Wellcome Trust Sanger Institute in Hinxton, Cambridge, UK. This registry can be approached at the website: www.sanger.ac.uk/ Software/Rfam/mirna. Queries can be sent to the e-mail address: microrna@sanger.ac.uk. Plant differentiation and microRNAs. One of the most elaborate systems of plant patterning is the differentiation of oral members. This system has been actively studied for about 15 yr and early updates of it were published by Schwarz-Sommer et al. (1990) and Coen and Meyerowitz (1991). The function of many genes (probably close to 100) was found to be implicated in oral patterning and most of these are encoding transcription factors. To fully appreciate the roles of microRNAs in this patterning a good knowledge of the molecular genetics of oral development, especially in Arabidopsis and snapdragon, is required. For a good review and source of pertinent literature, the readers are referred to the review by Jack

RNA SILENCING IN PLANTS

121

(2004). I shall provide a couple of examples on the involvement of microRNAs in ower patterning. Xuemei Chen (2004) of the Waksman Institute, Rutgers University, New Jersey, focused on APETALA2 (AP2) which is a class A gene involved in the identity of sepals in Arabidopsis. She found that miR172 is highly complementary to a region in AP2 and thus could be involved in ower patterning. She also found that in some mutants that are defective in the generation of microRNAs (e.g., hen1 and dcl-1) there was a several-fold higher level of the AP2 protein than in wild-type owers. Enhancing the expression of AP2 caused homeotic phenomena (aberrant owers). It thus seemed that miR172 keeps AP2 at low levels. Such a role of reducing the levels of specic proteins at proper times and locations is typical for microRNAs. Support for such a role was actually furnished by an independent study of Aukerman and Sakai (2003) who studied AP-2-like genes that affect owering time. One of these genes, EAT, had a non-coding sequence and this sequence had similarity to miR172. On the other hand, the target sites of miR172 were found in several AP2 family members in species other than Arabidopsis (e.g., TOE1, TOE2, TOE3) as well as in AP2 itself. These authors suggested that there is a gradual build-up of the miR172 level until, at a certain level, the proteins encoded by the AP2-like genes are reduced sufciently to permit oral initiation. Another phenomenon that appears to be regulated by microRNAs is the patterning of plant leaves. Experimental approaches to study leaf development started about 50 yr ago. In early studies surgical methods were applied to leaf initials in the shoot apex (e.g., Sussex, 1954; Snow and Snow, 1959). Clearly the dorsoventrality of leaves was disrupted by surgical incisions between the dome of the shoot apical meristem (SAM) and the incipial leaf primordium. Further studies increased knowledge on leaf differentiation and these were reviewed by Rob Martienssen and associates (Byrne et al., 2001). I shall focus on some Arabidopsis genes that are involved in leaf differentiation and in which microRNAs are probably involved. PHABULOSA is such a gene. The mutant phb-1d features adaxialization of leaves, with additional auxiliary meristems at the leaf base (adaxial means facing towards the shoot; abaxial means facing away from the shoot). PHAVOLUTA is a similar gene and in the Arabidopsis mutants phb and phv there is a dramatic transformation of leaf symmetry. John Bowman, Kathryn Barton and associates (McConnell et al., 2001) studied this change in leaf symmetry (conversion of abaxial leaf fate to adaxial fate). For example, the PHB gene has many introns and in some mutants (e.g., phb-1d) there was a change in one base that caused interference with normal splicing. The normal distribution of the PHB protein is consequently changed. Leaf symmetry and its control by class III HC-ZIP and KANADI genes was further studied by the team of Bowman (Emery et al., 2003). The gain of function of Phb and Phv (class III HC-ZIP family genes) and the loss of function of KANADI caused adaxilization of leaves in Arabidopsis. A gain of function of another gene (REVOLUTA) caused an alteration of the radial pattern of the vascular bundles in the shoot. When the coding sequence of the Rev gene was changed in a way that bases were changed without changing the encoded amino acid sequence, this caused a marked morphological change. Emery et al. (2003) suggested that this base change disrupted the capability of the respective mRNA for being a proper target to a specic microRNA. A further support for this suggestion resulted from the study of Mallory et al. (2004) who examined the effect of changing the coding sequence for Phb so that

the respective mRNA will not serve as a target for microRNAs (miR165/166). These and additional studies in this line seem to indicate that specic microRNAs lead the RISC-like particles to a specic mRNA target and by that reduce the product of this mRNA. For a better understanding of the microRNA/mRNA interaction in dorsoventral symmetry in plant differentiation, here are some more details. The short sequences of the mature microRNAs are target specic, meaning that minor changes in the nucleotide sequence in their target (the mRNA) can prevent the proper binding of the microRNA to its target. Several investigators used this target specicity because the sequence of the target (mRNA) can be changed without changing its coding. For example, a change from the triplet GGU to GGA will be a silent mutation: both these triplets code for glycine. But, a mature microRNA may be sensitive to such a U to A change and thus not bind to the mutated mRNA. Hence the mature microRNA will not guide the RISC-like particle to the mRNA. If it does, then a mutation in a transcription factor, such as PHB or PHV, is of this silent type. Could it be that these mutations encode the normal transcription factors but their respective mRNAs are not targets for the microRNAs that normally guide the RISC-like particle to these mRNAs? The team of Kathryn Barton (Bao et al., 2004) provided convincing evidence that this is indeed what happens with the phb-1d and phv-1d mutants. Moreover, Bao et al. (2004) also showed that in the wild-type PHB and PHV, coding sequences are heavily methylated downstream of the microRNA complementary strand and in phb-1d and phv-1d this methylation is reduced. The authors thus suggested a novel model of silencing: the microRNA interacts with nascent, newly processed PHB mRNA, to alter chromatin in the corresponding PHB template DNA, predominantly in differentiated cells. If this model is veried, the control of differentiation (e.g., dorsoventrality) will expand to TGS. In a preview for the Bao et al. (2004) publication, Eshed and Bowman (2004) noted that the kind of gene silencing reported by the former publication was previously found only for siRNA but not for microRNA. The few examples on the involvement of microRNA in the patterning of plants as well as several recent additional studies in this eld already indicate clearly that microRNAs play a vital role in this patterning. Moreover, the role of microRNAs is most probably not limited to morphological patterning. Changes in microRNA expression were revealed after plants were exposed to certain environmental conditions such as those causing stresses. Moreover, certain miRs were revealed only in stressed plants. It is therefore plausible that the vital role of microRNAs is not limited to structural differentiation but is also essential for normal plant metabolism. References
Ambros, V.; Bartel, B.; Bartel, D. P.; Burge, C. B.; Carrington, J. C.; Chen, X.; Dreyfuss, G.; Eddy, S. R.; Grifths-Jones, S.; Marshall, M.; Matzke, M.; Ruvkun, G.; Tuschl, T. A uniform system for microRNA annotation. RNA 9:277 279; 2003. Aukerman, M. J.; Sakai, H. Regulation of owering time and oral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15:27302741; 2003. Bao, N.; Lye, K.-W.; Barton, M. K. MicroRNA binding sites in Arabidopisis class III HD-ZIP mRNAs are required for methylation of the template chromosome. Dev. Cell 7:653662; 2004. Bartel, B.; Bartel, D. P. MicroRNAs: at the root of plant development. Plant Physiol. 132:709717; 2003.

122

GALUN MicroRNAs and their targets, including a stress-induced miRNA. Mol. Cell 14:787 799; 2004. Jorgensen, R. A. Sense cosuppression in plants: past, present and future. In: Hannon, G. J., ed. RNAi a guide to gene silencing. New York: Cold Spring Harbor Laboratory Press; 2003:521. Kusaba, M.; Miyahara, K.; Lida, S.; Fukuoka, H.; Takano, T.; Sassa, H.; Nishimura, M. Low glutelin content1: a dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice. Plant Cell 15:14551467; 2003. Lindbo, J. A.; Silva-Rosales, L.; Proebsting, W. M.; Dougherty, W. G. Induction of a highly specic antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5:17491759; 1993. Lippman, Z.; Martienssen, R. The role of RNA interference in heterochromatic silencing. Nature 431:364370; 2004. Liu, Q.; Singh, S. P.; Green, A. G. High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol. 129:1732 1743; 2002. Llave, C.; Kasschau, K.; Rector, M. A.; Carrington, J. C. Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:16051619; 2002a. Llave, C.; Xie, Z.; Kasschau, K. D.; Carrington, J. Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:20532056; 2002b. Longstaff, M.; Brigneti, G.; Boccard, F.; Chapman, S.; Baulcombe, D. Extreme resistance to potato virus-X infection in plants expressing a modied component of the putative viral replicase. EMBO J. 12:379386; 1993. Mallory, A.; Dugas, D. V.; Bartel, D. P.; Bartel, B. MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and oral organs. Curr. Biol. 14:10351046; 2004. Matzke, M. A.; Primig, M.; Trnovsky, J.; Matzke, A. J. M. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 8:643649; 1989. McConnell, J. R.; Emery, J.; Eshed, Y.; Bao, N.; Bowman, J.; Barton, M. K. Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411:709713; 2001. Meister, G.; Tuschl, T. Mechanism of gene silencing by double-stranded RNA. Nature 431:343349; 2004. Mette, M. F.; Van der Winden, J.; Matzke, M. A.; Matzke, A. J. M. Short RNAs can identify new candidate transposable element families in Arabidopsis. Plant Physiol. 130:69; 2002. Metzlaff, M.; ODell, M.; Cluster, P. D.; Flavell, R. B. RNA-mediated RNA degradation and chalcone synthase A silencing in Petunia. Cell 88:845854; 1997. Mueller, E.; Gilbert, J.; Davenport, G.; Brigneti, G.; Baulcombe, D. C. Homology-dependent resistance transgenic virus-resistance in plants related to homology-dependent gene silencing. Plant J. 7:10011013; 1995. Napoli, C.; Lemieux, C.; Jorgensen, R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279289; 1990. Ogita, S.; Uefuji, H.; Yamaguchi, Y.; Koizumi, N.; Sano, H. RNA interference producing decaffeinated coffee plants. Nature 423:823; 2003. Palauqui, J.-C.; Elmayan, T.; Pollien, J.-M.; Vaucheret, H. Systemic acquired silencing: transgene-specic post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 16:47384745; 1997. Park, W.; Li, J.; Song, R.; Messing, J.; Chen, X. CARPEL FACTORY, a dicer homolog and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12:14811495; 2002. Reinhart, B. J.; Weinstein, E. G.; Rhoades, M. W.; Bartel, B.; Bartel, D. P. MicroRNAs in plants. Genes Dev. 16:1616 1626; 2002. Roth, B. M.; Pruss, G. J.; Vance, V. B. Plant viral suppressors of RNA silencing. Virus Res. 102:97108; 2004. Ruiz, M. T.; Voinnet, O.; Baulcombe, D. C. Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937 946; 1998. Schwarz-Sommer, Z.; Huijser, P.; Nacken, W.; Saedler, H.; Sommer, H.

Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281297; 2004. Bass, B. L. Double-stranded RNA as a template for gene silencing. Cell 101:235238; 2000. Baulcombe, D. C.; English, J. J. Ectopic pairing of homologous DNA and post-transcriptional gene silencing in transgenic plants. Curr. Opin. Biotechnol. 7:173180; 1996. Bonner, J.; Huang, R.-C.; Maheshwari, S. The physical state of newly synthesized RNA. Proc. Natl Acad. Sci. USA 47:15481554; 1961. Byrne, M.; Timmermans, M.; Kidner, C.; Martienssen, R. Development of leaf shape. Curr. Opin. Plant Biol. 4:3843; 2001. Chen, X. A microRNA as a translational repressor of APETALA2 in Arabidopsis ower development. Science 303:20222025; 2004. Coen, E. S.; Meyerowitz, E. M. The war of the whorls genetic interactions controlling ower development. Nature 353:3137; 1991. de Haan, P.; Gielen, J. J. L.; Prins, M.; Wijkamp, I. G.; Vanschepen, A.; Peters, D.; Vangrinsven, M. Q. J. M.; Goldbach, R. Characterization of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants. Bio-Technology 10:11331137; 1992. Depicker, A.; Van Montagu, M. Post-transcriptional gene silencing in plants. Curr. Opin. Cell Biol. 9:373382; 1997. Ding, S. W.; Li, H. W.; Lu, R.; Li, F.; Li, W. X. RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res. 102:109115; 2004. Ding, S. W.; Li, W. X.; Symons, R. H. A novel naturally-occurring hybrid gene encoded by a plant RNA virus facilitates long-distance virus movement. EMBO J. 14:57625772; 1995. Emery, J. F.; Floyd, S. K.; Alvarez, J.; Eshed, Y.; Hawker, N. P.; Izhaki, A.; Baum, S. F.; Bowman, J. L. Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI Genes. Curr. Biol. 13:1768 1774; 2003. English, J. J.; Mueller, E.; Baulcombe, D. C. Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8:179188; 1996. Eshed, Y.; Bowman, J. L. MicroRNAs guide asymmetric DNA modications guiding asymmetric organs. Dev. Cell 7:629630; 2004. Fagard, M.; Boutet, S.; Morel, J.-B.; Bellini, C.; Vaucheret, H. AGO1, QDE2, RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc. Natl Acad. Sci. USA 97:1165011654; 2000. Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Potent and specic genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806811; 1998. Galun, E. Transposable elements a guide to the perplexed and the novice. Dordrecht: Kluwer Academic Publishers; 2003. Galun, E. RNA silencing. Singapore: World Scientic Publishing Co.; 2005. Galun, E.; Breiman, A. Transgenic plants. London: Imperial College Press; 1997. Galun, E.; Galun, E. Manufacture of medical and health products by transgenic plants. London: Imperial College Press; 2001. Golemboski, D. B.; Lomonossoff, G. P.; Zaitlin, M. Plants transformed with a tobacco mosaic-virus nonstructural gene sequence are resistant to the virus. Proc. Natl Acad. Sci. USA 87:6311 6315; 1990. Hamilton, A. J.; Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950952; 1999. Han, M. H.; Goud, S.; Song, L.; Fedoroff, N. The Arabidopsis doublestranded RNA-binding protein HYL1 plays a role in microRNAmediated gene regulation. Proc. Natl Acad. Sci. USA 101:10931098; 2004. Helliwell, C.; Waterhouse, P. Constructs and methods for high-throughput gene silencing in plants. Methods 30:289295; 2003. Horiguchi, G. RNA silencing in plants: a shortcut to functional analysis. Differentiation 72:6573; 2004. Huang, R.-C.; Bonner, J. Histone, a suppressor of chromosomal RNA synthesis. Proc. Natl Acad. Sci. USA 48:12161222; 1962. Jack, T. Molecular and genetic mechanisms of oral control. Plant Cell 16:S1S17; 2004. Jacob, F.; Monod, J. Genes de structure et gene de regulation dans la biosynthese de proteins. C.R. Biol. 249:12821284; 1959. Jones-Rhoades, M. W.; Bartel, D. P. Computational identication of plant

RNA SILENCING IN PLANTS Genetic-control of ower development by homeotic genes in Antirrhinum majus. Science 250:931936; 1990. Schweizer, P.; Pokorny, J.; Schulze-Lefert, P.; Dudler, R. Double-stranded RNA interferes with gene function at the single-cell level in cereals. Plant J. 24:895903; 2000. Segal, G.; Song, R. T.; Messing, J. A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 165:387 397; 2003. Smith, H. A.; Swaney, S. L.; Parks, T. D.; Wernsman, E. A.; Dougherty, W. G. Transgenic plant-virus resistance mediated by untranslatable sense RNAs expression, regulation, and fate of nonessential RNAs. Plant Cell 6:1441 1453; 1994. Smith, N. A.; Singh, S. P.; Wang, M.-B.; Stoutjesdijk, P. A.; Green, A. G.; Waterhouse, P. M. Total silencing by intron-spliced hairpin RNAs. Nature 407:319320; 2000. Snow, M.; Snow, R. The dorsoventrality of leaf primordia. New Phytol. 58:188207; 1959. Stoutjesdijk, P. A.; Singh, S. P.; Liu, Q.; Hurlstone, C. J.; Waterhouse, P. A.; Green, A. G. hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efcient and stable silencing. Plant Physiol. 129:1723 1731; 2002. Sussex, I. M. Experiments on the cause of dorsoventrality in leaves. Nature 174:351 352; 1954. Tang, G.; Galili, G. Using RNAi to improve plant nutritional value: from mechanism to application. Trends Biotechnol. 22:463469; 2004. Tang, G.; Reinhart, B. J.; Bartel, D. P.; Zamore, P. D. A biochemical framework for RNA silencing in plants. Genes Dev. 17:4963; 2003. van der Krol, A.; Mur, L. A.; Beld, M.; Mol, J. N. M.; Stuitje, A. R. Flavonoid genes in petunia: addition of a limited number of gene copies

123

may lead to a suppression of gene expression. Plant Cell 2:291299; 1990. Vance, V. B. Replication of potato virus-X RNA is altered in coinfections with potato virus-Y. Virology 182:486494; 1991. Vance, V. B.; Berger, P. H.; Carrington, J. C.; Hunt, A. G.; Shi, X. M. 50 -proximal potyviral sequences mediate potato-virus-X potyviral synergistic disease in transgenic tobacco. Virology 206:583590; 1995. Vargason, J. M.; Szittya, G.; Burgyan, J.; Hall, T. M. T. Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115:799811; 2003. Voinnet, O.; Baulcombe, D. C. Systemic signalling in gene silencing. Nature 389:553; 1997. Wassenegger, M.; Heimes, S.; Riederl, L.; Sanger, H. L. RNA-directed denovo methylation of genomic sequences in plants. Cell 76:567 576; 1994. Waterhouse, P. M.; Graham, M. W.; Wang, M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl Acad. Sci. USA 95:1395913964; 1998. Wesley, S. V.; Helliwell, C. A.; Smith, N. A.; Wang, M.; Rouse, D. T.; Liu, Q.; Gooding, P. S.; Singh, S. P.; Abbott, D.; Stoutjesdijk, P. A.; Robinson, S. P.; Gleave, A. P.; Green, A. G.; Waterhouse, P. M. Construct design for efcient, effective and high-throughput gene silencing in plants. Plant J. 27:581590; 2001. Ye, M. K.; Malinina, L.; Patel, D. J. Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426:874878; 2003. Zamore, P. D. Plant RNAi: How a viral silencing suppressor inactivates siRNA. Curr. Biol. 14:R198 R200; 2004.